Project Summary/Abstract Motility is critical to the life cycle and pathogenicity of many parasites. While targeting motility has been successful for the treatment of multiple bacterial diseases, the motility and motile structures of eukaryotic pathogens remain understudied and under exploited as a treatment targets. A bending wave that primarily propagates from the tip to the base of their flagellum drives the motility of pathogenic kinetoplastids, which are eukaryotic parasites that cause multiple neglected tropical diseases. This is unlike nearly all other eukaryotes, which beat from the base to the tip. Because kinetoplastid flagellum bending wave propagation direction switches under certain chemical and environmental conditions, and because the motile elements of kinetoplastid the flagellum are nearly identical to all other eukaryotes, it is likely that unique coordination mechanisms innate to axonemal dyneins, the molecular motors that drive flagellar motility, dictates this tip-to-base motility. Testing this hypothesis requires quantitative single-molecule biophysical characterization of kinetoplastid dynein coordination mechanisms. The broad goal of this research program is to enable the development of novel treatments for kinetoplastid- associated diseases that target the tip-to-base motility of kinetoplastid flagella. The specific aims of this project are to biophysically and biochemically characterize axonemal dynein from Trypanosoma brucei brucei, which will be used as a model for kinetoplastid flagella, and to identify trypanosome axonemal dynein regulation mechanisms that could yield tip-to-base motility. This interdisciplinary project will take molecular biological (RNAi, cloning, protein tagging), biochemical (ion exchange chromatography, in vitro reconstitutions, ATPase assays), and biophysical (ultrafast dual-trap optical tweezers, total internal reflectance fluorescence microscopy) experimental approaches. The collected data will be integrated and understood by making multi-scale quantitative biophysical models of flagellar waveform propagation direction. The expected outcome will a quantitative framework from which to develop pan-kinetoplastid drugs that target parasite motility. Successful completion of the project will ultimately lead to a greater understanding of the fundamental mechanisms of pathogenic parasite motility and could lead to novel treatments for African sleeping sickness, Chagas disease, and leishmaniasis.